SUMMARY : Labyrinthula vitellina var. paciflica requires a steroid as growth factor. Cholesterol, A*-cholestenone, fucosterol, and p-sitosterol are active. When cholesterol is modified by (1) the C3$ hydroxyl group becoming trans to the C 10 methyl group, (2) esterification of the C 3 hydroxyl group, or (3) saturation of the C5, 6 double bond, the resulting compound is inactive. Modification of the side chain of cholesterol or cholestenone by the addition of a C24 ethyl or vinyl group does not destroy activity but unsaturation at C22, 23 does, as do the more marked changes found in diosgenin, deoxycorticosterone acetate, and methyltestosterone. Bile salts are inactive. The specificity of the requirement indicates that active steroids function as essential metabolites rather than solely as protective agents. Active steroids may be obtained by this micro-organism in nature from host plants or from diatoms and bacteria associated with the hosts.The isolation in pure culture of the labyrinthula strains by Watson (to be published) made possible the investigation of their nutritional requirements. One isolate, L. vitellina var. pacifzca, has been found to require a steroid for growth. Since such requirements are rare among micro-organisms, a detailed investigation of steroid specificity has been made.
MATERIALS AND METHODSStock cultures of isolates of L . macrocystis var. atlantica and L. minuta var. atlantica were maintained at 20' in tubes containing the semi-solid medium given in Table 1. The addition of 1.0 mg yo (w/v) of cholesterol rendered this medium suitable for the maintenance of L . vitellina var. pacifzca.The agar in this medium appears to be necessary as a physical support without which these organisms settle to the bottom of the tube, or cling to its sides, where they grow poorly. High viscosity methylcellulose (0.5 yo (wlv), Hercules Powder Company) gave fair results as an alternative supporting agent but did not form a sufficiently homogeneous gel to give consistent or maximal growth. The constant motion provided by a rotating apparatus constructed after Baker (1949) substituted for mechanical support and allowed the smallest strain ( L . minuta var. atlantica) to grow as well in a liquid as in an agar-containing medium. Tubes of liquid medium were placed in a rack held at an angle of 11.5" to the horizontal and rotated at 21 r.p.m. by this apparatus. This method of cultivation appears most promising for more critical studies